Lambda ZAP: Improved Strategies for Expression Library Construction and Use

Abstract

A strategy is presented for the efficient construction of λ ZAP genomic expression libraries. Procedures are described for the evaluation of the status of vector DNA at each stage of library construction to facilitate troubleshooting. Ligation of λ ZAP cohesive ends and preparation of the multiple cloning site were verified by restriction enzyme digestion of vector DNA. Sonication was a rapid way of producing random chromosomal fragments of a size range ideal for expression library construction. The advantages of cloning into the Not I site of the λ ZAP polylinker are discussed. The choice of this site eliminated the need to perform the methylation of chromosomal DNA, which is required when the conventional Eco RI site is used. This method also facilitates restriction mapping of cloned inserts. Genomic expression libraries were constructed using this approach for Synechococcus sp. PCC7942, Synechocystis sp. PCC6803, and Prochlorothrix hollandica. The utility of expression libraries and in vivo excision was demonstrated by verifying the identity of clones coding for Synechococcus sp. PCC7942 cytochrome f, since the correct reading frames of these cloned inserts were determined unambiguously.